Key abiotic and biotic determinants of occurrence …...Izydorczyk et al. 2013, Ecohydrology & Hydrobiology Increasing the efficiency of the buffer zone by incorporation of geochemical

Designed by M. Łapińska

Key abiotic and biotic determinants of occurrence

and toxicological imapct of cyanobacterial blooms

in a lowland dam reservoir of Sulejów, Poland

Joanna Mankiewicz-Boczek

Bia łko reporterowe (lucyferaza)

Substancje wywołuj ącestres oksydacyjny

Substancje stanowi ącewzorce obco ści

Substancje o charakterzetrwałych zanieczyszcze ń

środowiska

Substancje zaburzaj ąceszlaki endokrynne

NFKBRE

ARE

AHRE

GRE

Established in 1973

Max surface [m2] 23.80 mln

Average depth [m] 3.30

Volume [m3] 78.9 mln

Average flow for multi-year period [m3/s] 26.90

Average water retention time [day] 42

Sulejów Reservoir

Study site

N

cyanobacterial bloom near the dam, 2012

Used for retention & recreation

Serves as alternative source of drinking water for Łódź

aglomeration (till 2004 as main source drinking water)

Fot. A. Skowron

� IDENTIFICATION of key abiotic (physico-chemical, hydrological)

parameters affecting the development of toxic cyanobacterial

blooms

� METHODS ELABORATION for monitoring of toxic

cyanobacteria (application of molecular methods for risk

assessment and early warning system)

� IDENTIFICATION of the impact of biotic parameters

and interactions on the trail: cyanobacteria / cyanophages /

bacteria / cyanoabcterial toxins / other organisms

� ESTIMATION OF BIOLOGICAL POTENTIAL of cyanobacteria

and cyanotoxins (cellular biosensors for detection and evaluation

of novel mechanisms of noxious bioactivity of cyanobacteria

Cyanobacterial studies

from 1997 …

� OPTYMISATION of biological structure of Pilica river

floodplain for selfpurification enhancement and REDUCTION of

diffusive and point sources pollution in the Pilica basin

Cyanobacterial

monitoring

Spring/SummerDetermination of physico-chemical parameters includ ing nutrients concentration: P-PO4, TP (>0.1 mg/l*), N-NO 3, N-NH4, TN (>1.5 mg/l*) Chlorophyll a (> 10 µg/l**) Phytoplankton analysis

Occurrence of Microcystis, Planktothrix, Anabaena Detection of toxigenic (potentially toxic) strains of cyanobacteria

PCR amplification of mcy genes(polymerase chain reaction)

Occurrence of microcystinsApplication of screening tests:determination of microcystins concentration – ELISA(enzyme-linked immunosorbent assay)

determination of microcystins toxicity – PPIA (protein phosphatase inhibition assay)

Confirmation of microcystins if ELISA showed > 2.5 µg/l Quantitative and qualitative analysis of microcysti ns – HPLC (high performance liquid chromatography)

Transdisciplinary interpretation of results Following the first and second principle of Ecohydrology , the identification of cause-effect relationshipwith comparative studies of the lake/reservoir typology, hydrochemistry, phytoplankton diversity and water toxicityare fundamental for developing a strategy to reverse eutrophication .(Zalewski 2000; Wagner et al. 2009)

Note: * critical values for eutrophication recommended by OECD (1983); ** relatively low probability of adverse health effect recommended by WHO (2003)

Proposed integral procedure of microcystin-producin g cyanobacteria monitoring for bathing water qualit y

Mankiewicz-Boczek et al. in Chorus[ed.],

2012, Current approaches to Cyanotoxin risk

assessment, risk management and regulations

in different countries.

Mankiewicz-Boczek in Zalewski M., Urbaniak

M. [eds.] 2012. Adaptation of ecohydrological

system solutions and biotechnologies for

Africa.


Interaction between cyanobacteria – toxic genotypes – toxin production -

– reservoir hydrology conditions

1%toxic Microcystis

genotypes

70%toxic Microcystis

genotypes

Gągała et al., 2014, Microbial Ecology

Influence of environmental factors on toxigenic activity

and cyanobacterial toxicity

55 days 18 daysAverage water retentiontime:

1 µg/Laverage microcystins

concentration

1 µg/Laverage microcystins

concentration

Project NSC 0964/B/P01/2010/39


BLAST homology analysis indicated 90% similarity to g91 gene

described for cyanophage from the genera Myoviridae strain Ma-LMM01

14 ACCTAACCAGATTG 170 GCTGGAGTATTAGAGTTAMCAAG-AST-T--TCCTCTGTGCCCATCTCTAGCGGCGACCT 15130 ACATCAGCGTTCGTTTCGGCACTGTAGCCGGTGCAGCCCTCAWTATAGTAGAGGGTAATA 71

Detection of cyanophages (g91 gene) capable of degrading cyanobacterial cells

Interaction CYANOBACTERIA / CYANOPHAGES

Study supported by the National Science Centre,

project number UMO-2013/11/N/NZ8/00607

Spearman Rank Order

Correlation (p<0.05)

Cyanophages (g91)

2010 (n=9) 2013 (n=10)

Total Microcystis (16S rRNA) 0.82 0.68

Toxigenic Microcystis (mcyA) 0.82 0.75

Cyanophage g91

Phylogenetical analysis


Detection of bacteria capable of degrading microcystins

cyanobacterial hepatotoxins

BLAST homology analysis indicated 95% similarity to gene mlrA

described for bacteria from the genera Sphingopyxis sp. C1

and Stenotrophomonas sp.

1 CTCCTCCCACAAATCAGGACGAGCCCAATGGCCACGGCGAATTCSACGAAATCCCAAGGG 6061 CGCCACCCGAGCCCTGCAACCGTTGGGGCCCACTCGGCAGTGACGTTTACGCCCAGTTCG 120121 TTATGGATCGCGTGAATGAGCACGCCACCCCACATCGATCCACCGAGCTTGTTGCATACG 180181 AAGACAGCGATGTTGGTGCCGGCAATGAACCCCGGAGCGATAACGAATTGCTTGACAATA 240241 ACGCCCCAGGCCGCGCCAGGATCGCCGGAGAACAGTGTCGGCAGGTCGCGCGGCAAATGC 300301 CAAGCCCACCACATTATGCCGAGGATCGCCGCTGCGGTCAGGGGGTCAAACTTCTTCAGG 360361 AGCTGCGGCAGCGCAGAGCCGCGCCAGCCCAGTTCTTCGAGCAGCGGGCCAGGGCTGAGT 420421 AGCAGCGATGCTGCCAGCCATCACATAAATGGCGA 455

Bacteria mlrA

Phylogenetical analysis

Mankiewicz-Boczek et al. 2015; Open Life Sciences formerly Central European Journal of Biology

Interaction CYANOBACTERIA / OTHER BACTERIA

0

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15.05.2013 12.06.2013 19.06.2013 02.07.2013 10.07.2013 24.07.2013 07.08.2013 15.08.2013 11.09.2013 01.10.2013

Tresta

MC

s con

cen

tratio

n [µ

g/L]

Microcystis

tox

ige

nic

str

ain

s

(mcyA

)/P

ote

nti

al M

Cs

de

gra

de

rs

(mlrA

) [g

en

e c

op

y n

um

be

r p

er

µL]

mcyA Microcystis (395 pz) [gene copy number/µL] mlrA (120 bp) [gene copy number/µL] MCs [µg/L]


projects number 0964/B/P01/2010/39 and UMO – 2012/07/N/NZ8/00599

0

10

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Kon

trol

a -

wod

a de

styl

owan

a +

MC

-LR

Mie

szan

ina

no. 0

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-LR

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no. 0

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szan

ina

no. 3

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-LR

Los

sof

mic

rocy

stin

-LR

aft

eron

e w

eek

[%]


Detection of bacteria capable of degrading microcystins

cyanobacterial hepatotoxins

Interaction CYANOBACTERIA / OTHER BACTERIA

phylogenetically identified colonies of bacteria, from which tested mixtures were created

Co

ntr

ol-

MC

-LR

wit

ho

ut

ba

cte

ria

Mix

no

. 1 +

MC

-LR

Mix

no

. 2 +

MC

-LR

Mix

no

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-LR

Mix

no

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no

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no

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no

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Mix

no

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Mix

no

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-LR

Mix

no

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+ M

C-L

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Mix

no

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+ M

C-L

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no

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+ M

C-L

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Mix

no

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+ M

C-L

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no

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+ M

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no

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no

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. 28

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. 29

+ M

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no

. 30

+ M

C-L

R

Mix

no

. 31

+ M

C-L

R

Mix

no

. 32

+ M

C-L

R

Degradation efficiency 0.6 µg/mL/day

Mankiewicz-Boczek et al. 2015; Open Life Sciences formerlyCentral European Journal of Biology

64 AGCGGCGGACGGGTGAGTAATGCCTGGGGATCTGCCCAGTCGAGGGGGATAACTACTGGA 123

124 AACGGTAGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCG 183

184 CGATTGGATGAACCCAGGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGAC 243

244 GATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACT 303

304 CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGC 363

364 CGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGTTGGT 423

424 AGCTAATAACTGCCAGCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCA 483

484 GCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCAC 543

544 GCAGGCGGTTGGATAAGTTAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTAA 603

604 AACTGTCCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG 663

664 TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAG 723

724 GTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT 783

784 GTCGATTTGGAGGCTGTGTCCTTGAGACGTGGCTTCCGGAGCTAACGCGTTAA-TCGACC 842

843 GCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGC 902

903 GGTGGAGCATGTGGTTTAATTCGATGCAACGCGAARAACCTTACCTGGCCTTGACATGTC 962

963 TGGAATCCTGTAGAGATRCGGGAGTGCCTTCGGGAATCAGAACACAGGTGCTGCATGGCT 1022

BLAST homology analysis indicated ≥94%

similarity to gene 16S rRNA described for bacteria

Aeromonas veronii w-s-03


projects number 0964/B/P01/2010/39 and UMO – 2012/07/N/NZ8/00599


ESTIMATION OF BIOLOGICAL POTENTIAL of cyanobacteria and cyanotoxins

1. The activity of NFKBRE Hep3B cell line was

different for crude and purified

cyanobacterial extracts.

2. Response of NFKBRE Hep3B cell line was

similar despite different microcystins

concentration in extracts (both in case of

crude and purified extracts).

3. Crude cyanobacterial bloom extracts

contained other than microcystins

metabolites, which activated cellular

biosensor NFKBRE based on Hep3B cell

line.

90

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(fo

ld o

f co

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lue

)

Concentration [ppm]

NFKBRE Hep3B, crude cyanobacterial extracts

CME1

CME2

CME3

467 µg MC/ml

191 µg MC/ml

21 µg MC/ml

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110

0 500 1000 1500 2000

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)

Concentration [ppm]

NFKBRE Hep3B, purified toxin preparations

PME1

PME2

PME3

268 µg MC/ml

94 µg MC/ml

18 µg MC/ml

Pilote study – Comparison of influence of purified and

crude cyanobacterial extracts on pattern recognition

receptor

Conclusions:


project number UMO – 2012/07/B/NZ8/03991


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Z1 Z2 Z3 Z4 Z5

Pho

spha

tes

conc

entr

atio

n[m

g/l] 3.09.2010

30.09.20103.11.20109.12.201019.01.2011

Critical level for

occurence of

cyanoabcterial blooms

GEOCHEMICAL BARRIER

Elaboration of system solutions

(www.geoportal.gov.pl)

Demonstration zone in Zarzęcin:

reduction of groundwater POLLUTION WITH PHOSPHORUS COMPOUNDS,

by strengthening the plant ecotone zone with geochemical barrier based on limestone.

www.ekorob.pl

(fot. EKOROB)


Izydorczyk et al. 2013, Ecohydrology & Hydrobiology

Increasing the efficiency of the buffer zone

by incorporation of geochemical barrier

www.ekorob.pl

Pho

spha

tes

conc

entr

atio

nin

gr

ound

wat

er[m

gPO

4/l]

przed za0

2

4

6

8

10

12

14

befor barrier after barrier

lowconcentrationof phosphatesin groundwater

high concentrationof phosphatesin groundwater

groundwater levelreservoir river

(fot. EKOROB)

(fot. EKOROB)

European Regional Centre for Ecohydrology PAS

Prof. dr hab. Joanna Mankiewicz-Boczek

Dr Katarzyna Izydorczyk

Dr Ilona Gągała

Mgr Aleksandra Jaskulska

Dep. of Applied Ecology, University of Lodz

Prof. dr hab. Joanna Mankiewicz-Boczek

Dr Tomasz Jurczak

Institute of Medical Biology PAS, Łódź

Prof. dr hab. Jarosław Dziadek

Dr hab. Łukasz Pułaski

Dr Dorota Jaros

Dr Jakub Pawełczyk

Mgr inż. Iwona Karwaciak

Department of Hydrobiology, Faculty of Biology,

Adam Mickiewicz University, Poznań

Dr hab. Mikołaj Kokociński

Dziękuję za uwagę ! Thank you for your attention !

Gracias por su atención!

Documents

Key abiotic and biotic determinants of occurrence …...Izydorczyk et al. 2013, Ecohydrology & Hydrobiology Increasing the efficiency of the buffer zone by incorporation of geochemical